Pre-stained 3,3&#39;,5,5&#39;-tetramethylbenzidine substrates for the detection of enzyme activity

ABSTRACT

The present invention relates to a method of using a storage-stable, pre-stained 3,3′,5,5′-tetramethylbenzidine (TMB) substrate in an enzyme substrate. The TMB substrates comprise: (a) a water-based TMB substrate composition which includes a peroxide, e.g., hydrogen peroxide or urea hydrogen peroxide, and (b) a dye which is soluble therein. Suitable dyes to be included in the substrate preferably have an absorbance at 450 nm of at the most milli absorbance units. Examples of dues are Pholixine B, Eosin B and Quinaldine Red. The substrates are advantageous in that they include a dye which is visible to the human eye. This is especially valuable when the substrates are handled and measured. The substrates are especially suitable for enzyme assays such as enzyme-linked-immunosorbent-assays (ELISA), e.g. horse radish peroxide (HRP) is used. The substrates may advantageously include less than 5% organic solvents and a solubility increasing agent such as polyvinylalcohol. The substrates are storage-stable for more than 12 months.

FIELD OF THE INVENTION

The present invention relates to 3,3′,5,5′-tetramethylbenzidine reagentsespecially for use in enzyme immunoassay techniques with enzyme labelledantibodies or antigens.

BACKGROUND OF THE INVENTION

Enzyme immunoassay (EIA) is an assay technique based on two facts: (1)the immune system of vertebrates can produce an almost unlimited varietyof antibodies each with a specific affinity for an antigen, and (2) thehigh catalytic power and specificity of enzymes, which it often is quiteeasy to detect. In the EIA technique, the immuno-reactants, the antibodyand the corresponding antigen are reacted with each other and thisreaction is detected using enzymes labelled to one of theimmuno-reactants. Different enzymes are used in the assay and amongthese are β-galactosidase, peroxidase, e.g. horseradish peroxidase, andalkaline phosphatase. For the detection of the enzyme activity is used achromogenic substrate and a chromogen which develop a colour during thereaction. This technique is very useful both for diagnostic purposes andin basic research work. The enzyme immunoassays have several advantages.It is possible to obtain very high sensitivity and specificity withrelatively cheap reagents and equipment. The methods to producemonoclonal antibodies have enhanced the possibility of standardisationof EIA with even higher sensitivity and specificity and contributes tonew assay designs.

A modification of the EIA and one of the very widely used enzymeimmunoassay techniques today is the ELISA(enzyme-linked-immunosorbent-assay) which can be used for bothqualitative and quantitative assays. In principle an enzyme is coupledto an antibody against the antigen to be determined. The assay isusually performed in trays of polystyrene or polypropylene to which theantigen is immobilised. The enzyme is chemically coupled to the antibodyand incubated with a substrate solution containing a suitable buffer, achromogenic substrate, e.g. hydrogen peroxide or urea peroxide, and achromogen which changes colour when the peroxide is oxidised. There aremany variations of an ELISA. In competitive assays, the enzyme may becoupled with the antigen. The number of “layers” of antigen andantibodies may vary as well as it is possible to use different enzymes.In ELISA techniques, horseradish peroxidase (HRP) is a commonly usedenzyme. For the detection of the enzyme activity is needed a specificenzyme substrate for the actual enzyme. Various enzyme substrates suchas ortho-phenylenediamine (OPD),2,2′-azino-bis-(3-ethylbenz-thiazoline-6-sulfonic acid) (ABTS) and3,3′,5,5′-tetramethylbenzidine (TMB) are commonly used. TMB is anon-hazardous normally colourless substrate, which forms an intenselyblue coloured oxidation product in the presence of peroxide and HRP. TMBis often preferred as substrate because it is at this time the mostsensitive substrate for HRP and is less toxic than e.g. OPD.

When performing an ELISA assay it is important to reduce the backgroundsignal in order to obtain the best possible sensitivity. It hastherefore been considered important that the substrate componentsthemselves should be colourless or only very slightly coloured in orderfor them to be used as substrate in an ELISA assay. Such properties willminimise the blind absorbance values. This means that pipetting of thethus colourless substrate into the enzyme immunoassay reaction vessel,which often is a transparent tube or a transparent ELISA tray, isassociated with the risk of incorrect dosage in that it may be difficultto determine immediately by visual examination whether the substrate hasbeen added to a vessel (e.g. a well) or not. As a consequence, thesubstrate may not be added to all vessels, or twice the required amountof substrate may be added to some vessels. In automated assays it is noteasily achievable to make control measurements to ensure that thesubstrate or the proper amount of substrate has been added.

German Patent No. DE 195 27 160 C1 describes a method forphotometrically measuring of the concentration of an enzyme by measuringa colour created by enzymatic reaction with a colourless substrate. Theenzymatic reaction is stopped by adding 2 N NaOH. To control theaddition of stopping solution a dye (Amaranth) is included in theotherwise colourless solution. The German patent relates to theinclusion of a dye in a stop solution, but does not address the problemsof storage stability and construction of water-based substrate systemsif a dye should be included in such systems for the same purpose.

U.S. Pat. No. 4,128,629 describes a method for determining smallconcentrations of cortisol by using an immunoassay technique, morespecific a radio-immuno-assay (RIA). In an example the possibility ofincluding a non-interfering red dye to indicate the presence of thetracer used in the assay is mentioned. There is no mentioning of enzymeimmunoassays and the specific problems associated to the stabilisationand storage stability of substrate solutions having dyes includedtherein.

U.S. Pat. No. 5,318,894 describes a dry phase test device and method fordetermining the presence of a peroxidatively active substance. The testdevice includes a test pad comprising a suitable carrier matrix with anindicator reagent. The indicator reagent composition for impregnation ofthe test area of the test device includes an indicator dye (e.g. TMB), ahydroperoxide and a promoter. To improve the colour resolution anddifferentiation one or more inert background dyes can be included in theindicator reagent. The amount of organic solvent present in theindicator reagent for impregnation is up to 90%. U.S. Pat. No. 5,318,894does however not address the problems with respect to preparation andstorage stability of a water-based TMB substrate solution system.Storage stability of the liquid compositions does not appear to berelevant in that the liquid compositions are used immediately afterpreparation. Also, the dyes used in U.S. Pat. No. 5,318,894 appears tobe selected so that they deliberately have a high absorbance in the samerange as the reacted TMB substrate.

DESCRIPTION OF THE INVENTION

The present invention provides a solution to the problems associatedwith applying an otherwise colourless substrate to a reaction vessel(e.g. manual pipetting of a TMB substrate composition into an ELISAtray) by including a dye in ready-to-use substrates. In this way it hasbecome possible, e.g. to see the substrate both in the pipette and inthe reaction vessel, such as an ELISA tray, during pipetting. The dye isso selected that it has substantial no absorbance in the absorbancerange under the conditions where the reaction product of the reactionbetween the substrate and the enzyme is to be detected. Furthermore, theespecially selected dyes have substantially no influence on theenzyme-substrate reaction. It has therefore for the first time becomepossible to provide a substrate which makes manual pipetting easier andmore reliable, and thereby also faster without compromising the storagestability of the substrate.

It should be understood that the substrates according to the presentinvention are equally applicable for automatic pipetting procedureswhere it is desirable to control (either manually or by automatic means)the addition of a sufficient amount of substrate to the reaction vessel.

Thus generally, the present invention provides a substrate for use in anenzyme immunoassay (where the enzyme immunoassay includes the reactionbetween an enzyme and an enzyme substrate comprised in the substrate);the substrate comprises a dye which is (a) visible to the human eye whenapplied to the environment where the enzyme immunoassay is conducted,(b) substantially non-interfering in the reaction between the enzymesubstrate and the enzyme, and (c) substantially non-interfering underthe conditions used for measuring the reaction product of the reactionbetween the enzyme substrate and the enzyme.

The substrates described herein may be in the form of a powder, a tabletor a liquid formulation, preferably, the substrates are in the form of aliquid formulation, e.g. a ready-to-use solution, or in the form of atablet which may easily be dissolved in a suitable medium, e.g. water, abuffer, or a mixture of an organic solvent and water, to provide asolution having the desired concentration.

The substrates may be applicable for any enzyme immunoassays (EIA) knownto the person skilled in the art. Of the various EIA techniquesavailable, the ELISA technique is especially interesting since automatedset-ups are commercially available. For the HRP-ELISA, suitable enzymesubstrates are ortho-phenylenediamine (OPD),2,2′-azino-bis-(3-ethylbenz-thiazoline-6-sulfonic acid) (ABTS),diaminobenzidine (BAB), and 3,3′,5,5′-tetraalkylbenzidine such as3,3′,5,5′-tetramethylbenzidine (TMB), among which3,3′,5,5′-tetramethylbenzidine (TMB) is preferred due to itsnon-hazardous nature. (it should be understood that the presentinvention relates to comprising any and all of the above-mentionedenzyme substrates. If alkaline phosphatase is used as enzyme in the EIAsubstrates, the enzyme substrate may be one of5-bromo-4-chloro-3-indolyl phosphate (BICP), p-nitrophenyl phosphate(p-NPP), and Fast Red.

In particular, the present invention provides a storage-stablepre-stained 3,3′,5,5′-tetramethylbenzidine (TMB) substrate comprising:(a) a water-based TMB substrate composition which includes a peroxideand (b) a dye which is soluble therein.

When used herein, the terms “pre-stained substrate” and “substrates” andsimilar expressions are intended to mean a substrate compositioncomprising a dye.

The term “visible to the human eye” is intended to mean that thesubstrate in question is sufficiently coloured to be identified by thetechnician in the amounts typically used in enzyme immunoassays. Whenthe substrate is a TMB substrate where the reaction product between TMB,peroxide and HRP is measured at around 450 nm (yellow), it is preferredthat the substrate (dye) has a visible colour in the range of 500-600nm, i.e. in the range clear of the blue and the yellow colours of TMB.It is believed that the absorbance at a wavelength in that range(500-600 nm) should be at least 100 mabs, such as at least 200 mabs,preferably at least 400 mabs.

In order for a dye to be included in a substrate, e.g. in addition to analready established ready-to-use substrate such as a TMB substratecomposition, the dye has to fulfil certain criteria.

It is important that the dye is substantially non-interfering in thereaction between the enzyme substrate and the enzyme. Thus, in the casewhen a horse radish peroxidase-ELISA (HRP-ELISA) is performed, the dyeshould be substantially non-interfering in the reaction betweenperoxide, the horseradish peroxidase and TMB as well as substantiallynon-interfering with the reaction product. It should be understood thata small interference may be acceptable for certain applications,however, in general interference with the reaction may severelycompromise the sensitivity of the assay.

As a further criterion, the dye must be completely soluble under theconditions where the reaction product of the enzyme substrate/enzymereaction is measured. It is believed that dyes having a solubility inthe substrate of at least 5 μg/ml, preferably at least 10 μg/ml aresuitable for the purpose described herein. (When the substrate ispresented in the form of a powder or a tablet, the solubility of the dyerefers to the final (ready-to-use) concentration.)

A further but not less important criterion (which represents the gist ofthe present invention) is that the substrate (including the dye) shouldbe visible when it is applied to the reaction vessel where the enzymeimmunoassay is conducted, e.g. pipetted into the ELISA tray. However, inconnection with this important feature of the dye, it should besubstantially non-interfering under the conditions used for measuringthe reaction product of the reaction between the enzyme substrate andthe enzyme. This should apply even in the case where further reagents(e.g. stop-solutions) are added to the reaction vessel beforemeasurement of the reaction product is performed. In the case where theenzyme immunoassay is based on a HRP/TMB system, the dye should have noinfluence on the detection of the reaction product, especially not afteraddition of a stop-solution (e.g. sulphuric acid). Thus, the dye itselfshould not have any or very little absorbance at the measuringwavelength, often in the range of 400-500 nm or 600-660 nm, inparticular at around 450 nm and around 640 nm. For a normal TMBsubstrate (without dye) the usual blind absorbance values are from 5-20milli absorbance units (mabs; measured for a volume of 100 μl in a ELISAtray (diameter 6.7 mm) [this volume to diameter ratio applies generallyto the mabs values stated in the description and the claims (unlessotherwise stated)]). The dye may cause a small increase, however, thetotal blind absorbance level should preferably be below 100 mabs,preferably below 75 mabs, such as below 50 mabs at least around 450 nmand around 640 nm, but preferably also in the entire ranges of 400-500nm and 600-660 nm. Thus, dyes which change from coloured to colourlessor colours which have no absorbance at the measuring wavelength underthe assay condition can be used.

Since the dye preferably is included in a ready-to-use substratecomposition, any (negative) effect on the activity and the long termstability of the substrate, when stored at 4° C. and even at 25° C.,should be minimal. The long term stability of the substrate comprising adye should be as good as for the normal colourless substrate, i.e. oftenat least 12 months (such as 12-18 months or more) when kept at 4° C. inthe dark with less than a 25% decrease in the activity when compared tothe initial activity of the substrate. Preferably the reduction of theactivity is even less, such as less than 20%, or even less than 10% orless than 5%.

When used herein, the term “TMB substrate composition” is intended tomean an (otherwise substantially colourless) composition which may beused in its own right. Thus, when used herein, a substrate compositionmay be an already established substrate, however, this should not beconstrued so that the present invention is limited to inclusion of dyesin presently commercially available TMB substrate compositions.

TMB substrate compositions will typically comprise a number of additivesin order to enhance the stability and compatibility with the HRP-ELISAor HRP-EIA. Furthermore, the TMB substrate composition may furthercomprise the necessary peroxide so that the TMB substrate compositionconstitutes a “complete” substrate system. A peroxide as a part of theTMB substrate composition is typically selected from hydrogen peroxideand urea hydrogen peroxide, preferably hydrogen peroxide.

From the above it should be understood that the dye should not be asubstrate or an inhibitor for horseradish peroxidase (HRP) in the casewhere the substrate is used in a HRP-ELISA or a HRP-EIA.

In order to avoid hazardous conditions for the technicians working withthe substrates, it is especially preferred that the substrates accordingto the present invention comprises a TMB substrate which includes awater-base solvent system or a solvent system comprising less than 5%(v/v) of organic solvents. In the case where a water-based solventsystem is applied (but also in certain cases where an organic solventsystem is used) it may be necessary or desirable to include one or moresolubility increasing agent(s) in order to facilitate the (permanent)dissolution of the dye. An example of a suitable solubility increasingagent is polyvinyl alcohol, which may already be a constituent of theTMB substrate composition. It is obvious that the relatively low contentof organic solvents has been a further challenge towards a solution tothe problem solved with the present invention.

Examples of commercially available TMB substrate compositions are: “TMBOne Substrate” (ex Kem-En-Tec A/S, Denmark), “TMB PLUS Substrate” (exKem-En-Tec A/S, Denmark), “TMB Microwell Peroxidase Substrate” (exKirkegaard & Perry Laboratories), “K-Blue” (ex ElisaTechnologies) and“TMB Peroxidase Substrate” (ex MOSS Inc.).

For substrates for other enzyme immunoassays similar principles applymutatis mutandis.

In the present context the term “dye” is intended to have its normalmeaning, namely a “soluble” colorant. Dyes which have suitableproperties with respect to at least some of the above-mentionedparameters are, e.g., Phloxine B, Eosin B, and Quinaldine Red, NaphtholYellow, Basic Fuchsin, m-Cresol Purple, Thymol Blue, Xylenol Blue, NileBlue A, Thymolphthalein, among which Phloxine B seems to be the mostsuitable.

A preferred embodiment of the present invention, relates to a substratecomprising 3,3′,5,5′-tetramethylbenzidine (TMB), Phloxine B, hydrogenperoxide, one or more additives including any solubility increasingagent(s), and a water-based solvent system.

The present invention also relates to the use of the substratesdescribed herein in an enzyme immunoassay, such as an ELISA.

Furthermore, the present invention also relates to the use of a dye forthe preparation of substrate for an enzyme immunoassay, such as anELISA, where said dye is present in the substrate in such aconcentration that the dye is visible to the human eye. Especiallyinteresting dyes for that purpose are Phloxine B, Eosin B, andQuinaldine Red.

EXAMPLES

General Procedure for ELISA Assay Using TMB Substrates

In all of the following examples, the following ELISA procedure has beenused. The assay is a three layer sandwich ELISA. For all three layersare used Rabbit Anti-Human IgG, Human Serum Protein Calibrator, andRabbit Anti-Human IgG-HRP from Dako A/S, Denmark. The plate is coatedwith the first antibody to which the antigen is coupled as the secondlayer. This layer is usually the sample to be analysed but since our aimis to analyse the substrate the “sample” is the same antigen in allassays. To this antigen is coupled a second antibody-enzyme conjugate.The assay is in the following described in details.

The assay is performed in Polystyrene MAXISorp plates (flat bottomedwells) from Nalge NUNC International.

First layer: Rabbit Anti-Human IgG (DAKO A 0423); is diluted 1:1000 in0.1 M KH₂PO₄, pH 7.2, 100 μl is added to each well and incubatesovernight at 4° C. The antibody is decanted and the wells are washedonce with washing buffer (0.1 M KH₂PO₄; 0.5 M NaCl; 0.1% Tween 20; pH7.2). 200 μl blocking solution (0.1 M KH₂PO₄; 0.5% Tween 20; pH 7.2) isadded and incubates 30 minutes at room temperature. The blockingsolution is decanted and the wells washed twice with washing buffer.

Second layer: Human Serum Protein Calibrator (DAKO X 0908) is diluted to4 ng/ml in 0.1 M KH₂PO₄, pH 7.2, 100 μl is added to each well andincubates overnight at room temperature. The antigen dilution isdecanted and the wells washed three times in washing buffer.

Third layer: Rabbit anti Human IgG-HRP (DAKO P 214) is diluted 1:1000 in0.1 M KH₂PO₄, pH 7.2, 100 μl is added and incubates 2 hours at roomtemperature. The antibody enzyme-conjugate is decanted and the wellswashed three times in washing buffer.

The plate is then ready for addition of the TMB substrate to be tested.For each substrate to be tested is used one column (=eight wells). Thismeans that each substrate is analysed in an eight fold determination.100 μl substrate is added and incubates for 13 minutes at roomtemperature. The reaction is stopped by adding 100 μl 0.2 M sulphuricacid and the plate is read in an ELISA (BIO-TEK Instruments EL 312e) at450 nm with 630 nm as reference wavelength. When running an assay astable TMB Substrate, TMB Microwell Peroxidase Substrate from Kirkegaard& Perry Laboratories, is included as standard in one column in everytray. The activity of the tested substrate is the mean of eightdeterminations and is compared to this standard.

The blind absorbance value of the substrate is determined by adding 100μl substrate to a column of an uncoated ELISA plate adding 100 μl 0.2 Msulphuric acid and reading the plate at 450 nm with 630 nm as referencewavelength. The blind absorbance value is the mean value of eightdeterminations.

Example 1

Addition of Dye to TMB Substrates

Phloxine B (Sigma) is a dye which is red to purple at the pH of thesubstrate and change to colourless by addition of sulphuric acid.Phloxine B was added to 5 different ready-to-use substrates: TMB OneSubstrate; Kem-En-Tec A/S, TMB PLUS Substrate; Kem-En-Tec A/S, TMBMicrowell Peroxidase Substrate; Kirkegaard & Perry Laboratories (TMB 1),K-Blue; ElisaTechnologies (TMB 2) and TMB Peroxidase Substrate; MOSSInc. (TMB 3) The concentration of the dye was 15 μg per ml of substrate.The pre-stained substrates were visually examined for precipitation ofthe dye and graduated from “+++” (heavy precipitation) to “−” (noprecipitation). Furthermore, the blind value of the substrates was readat 450 nm with 630 nm as reference wavelength in an ELISA-tray, seegeneral procedure above. The results are shown in Table 1.

TABLE 1 Blind value Blind value Pre-stained Reference SubstratePrecipitation (mabs) (mabs) TMB One −  27 9 TMB Plus +++ — 8 TMB 1 − 1029 TMB 2 +++ — 47  TMB 3 − 74 9

For two of the substrates, TMB Plus and TMB 2, the addition of dyecaused heavy precipitation when the dye was added to the ready-to-usesubstrates. There was no precipitation in three of the remaining dyedsubstrates. One of them (TMB One) will be usable in an ELISA because theblind value is lower than the desired 50-75 mabs. For the other twosubstrates (TMB 1 and TMB 3), the addition of the dye caused an increasein the blind value to a level higher than or very close to 75 mabs.

Example 2

Improving the Solubility of the Dye

To improve the solubility, polyvinyl alcohol, which is one of thecomponents already present in some of the substrate compositions, wasadded. Polyvinyl alcohol (PVA) is present in the two Kem-En-Tecsubstrate compositions (TMB One and TMB Plus) and the amount wasincreased by adding 0.100 mg/ml and 0.152 mg/ml respectively to a finalconcentration of 0.200 mg/ml. To the other three tested substrates wasadded 0.200 mg PVA/ml to a concentration of at least 0.200 mg/ml.Subsequently, Phloxine B was added in the same concentration as inExample 1, i.e. 15 μg/ml.

The pre-stained substrates were again visually examined forprecipitation of the dye and the blind value of the substrates was readat 450/630 nm in an ELISA-tray (see the general procedure above). Theresults are shown in Table 2.

TABLE 2 Blind value Blind value Pre-stained Reference SubstratePrecipitation (mabs) (mabs) TMB One − 24 9 TMB Plus − 17 8 TMB 1 + — 9TMB 2 ++ — 47  TMB 3 − 67 9

TMB One and TMB Plus both had good visibility after pipetting in theELISA tray and acceptable blind values well below 50 mabs.

Example 3

Determination of the Optimal Concentration of the Dye

To the TMB Standard and TMB Plus substrates were added extrapolyvinylalcohol as in example 2 and Phloxine B was added in theconcentrations stated in Table 3.

TABLE 3 2 μg/ml 10 μg/ml 50 μg/ml Substrate Precip. Blind Precip. BlindPrecip. Blind TMB One − 23 − 29 + — pre-stained TMB Plus − 18 − 21 − 30pre-stained

The optimal concentration of dye is a combination of its solubility inthe substrate formulation, the increase in the blind value and thecolour must be strong enough to be visible in the ELISA tray. With aconcentration of 2 μg/ml the substrates are almost colourless. Aconcentration of 10 μg/ml gives good visibility and an acceptable blindvalue. The high concentration of 50 μg/ml leads to precipitation of dyein the TMB One substrate. In the TMB Plus substrate composition theblind value is below 50 mabs. Since the visibility is good at both 10and 50 μg/ml, it is p referred to add only the necessary amount of thedye and a concentration of 10 μg/ml thus seems to be sufficient.

Example 4

A Dyed TMB Substrate vs. a “Normal” TMB Substrate

The activity of TMB One substrate and TMB Plus substrate was compared tothe activity when extra PVA and a dye was added to the formulation.Extra PVA was added as in example 2. The dye Phloxine B was used in aconcentration of 10 μg/ml.

TABLE 4 Substrate Blind (mabs) Activity (mabs) TMB One 12  950 TMB One25  977 Pre-stained TMB Plus 19 1164 TMB Plus 32 1193 Pre-stained

These results show that the addition of a dye to the two TMBformulations has very little influence on the blind absorbance value andthe activity is the same for both substrates.

Example 5

Test of Various Dyes

Three different dyes Phloxine B, Eosin B and Quinaldine Red all fromSigma Chemical Co. were tested for their performance when included inthe formulation of TMB Plus substrate in a concentration of 10 μg/mltogether with polyvinylalcohol (0.152 mg/ml-total amount 0.200 mg/ml).The performance was tested as described in the general procedure. Theresults are shown in Table 5.

TABLE 5 Dye Blind (mabs) Activity (mabs) No dye (reference)  9 1083Phloxine B 15 1063 Eosin B 17  999 Quinaldine Red 17  843

None of the dyes precipitated at a concentration of 10 μg/ml. The blindvalue is good for all the selected dyes, but the activity is slightlyaffected by Eosin B and Quinaldine Red. The decrease in activity forEosin B is about 8% and could be acceptable but for Quinaldine Red thedecrease is about 25% which for some practical uses may be unacceptable.In certain cases it may, however, not be a prohibitive effect for theuse of the substrate and should therefore be regarded as being withinthe scope of the present invention.

Example 6

Long Term Stability of Pre-stained Substrates

To examine the long term stability of the pre-stained TMB Standard andTMB Plus substrate, small bottles (Nalgene amber 15 ml bottles)containing freshly produces substrate were placed at 4° C. and 20° C. inthe dark. From time to time bottles of substrate were analysed in theassay described in the general procedure. The activity of the substratewas set to 100% at the day of production. The results of the stabilitytests are shown in the tables below.

TABLE 6 TMB One Blind absorbance mabs Activity % Substrate 4° C. 20° C.4° C. 20° C. Day 1  19 (8)*  19 (8)*  100 (100)* 100 (100)* Day 84  30(13)* 29 (12)*  98 (100)*  99 (100)* Day 345 30 (19)* 33 (18)* 100(100)* 74 (77)* *( ) Reference value for unstained substrate

TABLE 7 TMB One Blind absorbance mabs Activity % Substrate 4° C. 20° C.4° C. 20° C. Day 1  25 (8)* 25 (8)* 100 (100)* 100 (100)* Day 105 23(7)* 26 (7)*  98 (100)*  64 (78)* Day 366 19 (9)*  40 (21)*  96 (100)* 33 (44)* *( ) Reference value for unstained substrate

As seen from the tables both substrates have very good long termstability, very similar tot he un-stained commercial substrates. Afteralmost a year at 4° C. the TMB Standard substrate did not lose anyactivity and there was no increase in the blind absorbance. At 20° C.there was loss in activity of about 25% which is generally expected fora substrate after storing at ambient temperature for that long time. Forthe TMB Plus substrate there was no increase in the blind absorbance andvery little decrease in activity at 4° C. (virtually insignificant).After around one year at 20° C. there was some loss in activity. It wasexpected observe a higher loss at 20° C. for this substrate due to thehigher activity level.

What is claimed is:
 1. A method of using a storage-stable, pre-stained3,3′,5,5′-tetramethylbenzidine (TMB) substrate in an enzyme immunoassay,where the enzyme immunoassay includes the reaction between an enzyme andTMB; said substrate comprising a water based TMB substrate compositionwhich includes a peroxide and a dye, said dye being (a) visible to thehuman eye when applied to the reaction vessel wherein the enzymeimmunoassay is conducted, (b) substantially non-interfering in thereaction between TMB and the enzyme, (c) substantially non-interferingunder the conditions used for measuring the reaction product of thereaction between TMB and the enzyme, and (d) completely soluble in thesubstrate under the conditions where the reaction product of TMB and theenzyme is measured.
 2. The method according to claim 1, wherein theactivity of the substrate decreases with less than 25% compared with theinitial activity when the substrate is stored in the dark for a periodof a least 12 months at 4° C.
 3. The method according to claim 2,wherein the decrease in activity is less than 20%.
 4. The methodaccording to claim 1, wherein the peroxide is selected from hydrogenperoxide and urea hydrogen peroxide, preferable hydrogen peroxide. 5.The method according to claim 1, wherein the substrate comprises lessthan 5% (v/v) of organic solvents.
 6. The method according to claim 1,wherein the solubility of the dye in the TMB substrate composition is atleast 10 μg/ml.
 7. The method according to claim 1, wherein thepre-stained TMB substrate has an absorbance at 450 nm of at the most 100milli absorbance units (mabs).
 8. The method according to claim 1,wherein the pre-stained TMB substrate has an absorbance in the entirerange of 400-500 nm of at the most 75 milli absorbance units (mabs). 9.The method according to claim 1, wherein the pre-stained TMB substratehas an absorbance at 640 nm of at the most 100 milli absorbance units(mabs).
 10. The method according to claim 1, wherein the pre-stained TMBsubstrate has an absorbance in the entire range of 600-660 nm of at themost 75 milli absorbance units (mabs).
 11. The method according to claim1, wherein the pre-stained TMB substrate has an absorbance at awavelength in the range of 500-600 nm of a least 100 milli absorbanceunits (mabs).
 12. The method according to claim 1, wherein the dye isnot a substrate or an inhibitor for horse-radish peroxidase (HRP). 13.The method according to claim 1, wherein the dye is not affecting thereaction or the reaction product from the reaction between3,3′,5,5′-tetramethylbenzidine (TMB), horse-radish peroxidase (HRP), anda peroxide.
 14. The method according to claim 1, wherein the dye isselected from Phloxine B, Eosin G, and Quinaldine Red.
 15. The methodaccording to claim 14, wherein the dye is Phloxine B.
 16. The methodaccording to claim 1, wherein the substrate further comprising one ormore solubility increasing agent(s).
 17. The method according to claim16, wherein the solubility increasing agent is polyvinylalcohol.
 18. Themethod according to claim 1, wherein the substrate comprises3,3′,5,5′-tetramethylbenzidine (TMB), Phloxine B, hydrogen peroxide, oneor more additives, and a water-based solvent system.
 19. The methodaccording to claim 1, wherein the enzyme immunoassay is an ELISA.